Activation of anti-oxidant Keap1/Nrf2 pathway modulates efficacy of dihydroartemisinin-based monotherapy and combinatory therapy with ionizing radiation.

The efficacy of radiotherapy depends not only on DNA damage but also on ROS production, both induced by ionizing radiation. Massive ROS production can induce cell death or activate protective pathways such as Keap1/Nrf2 pathway, which regulates intracellular cysteine availability through upregulation of SLC7A11, a subunit of xCT transporter, and subsequently glutathione synthesis, thus improving antioxidative defense. The anti-malaria drug dihydroartemisinin (DHA) shows anti-neoplastic potential. Previous publications suggested that DHA increased ROS production. We intended to enhance oxidative stress with DHA to improve the efficacy of radiotherapy. Therefore, we first analyzed the oxidative response to DHA in HCT116 colorectal and NCI-H460 lung adenocarcinoma cells. In response to DHA, we detected lipid peroxidation and protein oxidation, which resulted in mitochondrial damage and eventually in iron-dependent cell death. Concurrently, DHA activated Keap1/Nrf2 pathway in HCT116 cells, leading to increased SLC7A11 expression and glutathione level. In Keap1-mutant NCI-H460 cells, Nrf2 was constantly activated and responsible for high SLC7A11 and glutathione levels. Pancancer analysis revealed that lung cancer is the tumor entity with the most frequent Keap1 alterations. Although NCI-H460 cells reacted more refractory to DHA-induced cell death than HCT116 cells, eradication of clonogenic cells by DHA was more efficient in both cell lines when Keap1/Nrf2 pathway was inhibited. When applied simultaneously, radiotherapy and DHA more efficiently eradicated clonogenic cells than either therapy alone, but treatment schedule can mitigate the combinatory effect in HCT116 cells. In summary, DHA improved efficacy of radiotherapy, but treatment schedule must be considered with care especially in Keap1-wildtype cells.